SUMMARY The epidermal growth factor receptor (EGFR) is a model tyrosine kinase whose overexpression is common in various cancers, including basal-like breast cancer (BLBC). It is becoming increasingly clear that EGFR underlies not only conventional biochemical regulation induced by ligand-receptor binding but also much less well understood spatial and temporal regulation mechanisms. A heterogeneous distribution of the receptor at the plasma membrane results in an enrichment of EGFR in membrane regions with lateral dimensions of tens to hundreds of nanometers where receptor dimerization and oligomerization is favored due to a high local concentration of receptors. These clusters are not static but undergo continuous structural fluctuations. The relationship between this dynamic structure and the local signaling activation are insufficiently understood, partly because of a lack of appropriate optical tools for mapping subdiffraction limit dynamics with high temporal bandwidth and long observation time. Aim 1 of this application takes advantage of the unique photophysical properties of plasmonic nanoparticles (NPs) that provide large and stable optical signals and also encode information about deeply subdiffraction limit separations between NPs in their far-field spectrum to characterize the lateral diffusion and structural dynamics of individual EGFR clusters with high temporal resolution and without physical limitation in observation time. Previous implementations of plasmon coupling microscopy (PCM) utilized the NP spectrum detected under darkfield illumination to identify EGFR clustering. Dark-field detection requires, however, large (~40 nm) NP labels. In Aim 1, we will implement a new interferometric PCM (iPCM) for detecting 5 nm (to probe EGFR-EGFR contacts) and 10 nm (to probe EGFR oligomerization and clustering) gold NP labels. iPCM will be augmented with a correlation analysis to quantify continuous fluctuations in plasmon coupling. This technology will be applied to test the hypothesis that EGF binding results in a decrease of intracluster dynamics and an increase in EGFR phosphorylation. Another insufficiently understood element of structural regulation that is associated with EGFR clustering is lateral signal propagation through inter-receptor contacts or EGF-induced second messenger release. Aim 2 will elucidate how EGFR clustering impacts NP-EGF-induced reactive oxygen species (ROS) formation and ROS-mediated EGFR activation. In this approach EGF-functionalized NPs (NP- EGF) with known EGF loading are not simple imaging tools for mapping sub-diffraction limit clusters of EGFR, but instead, represent quantifiably units of local EGFR activation. In Aim 3 nanoconjugated EGF will be applied as probe to quantify the cross-talk between EGFR activation and nitric oxide (NO) formation. As NO synthesis is spatially and temporally strictly regulated, its concentration depends on EGFR activation, which varies as function of the local NP-EGF concentration. Aim 3 will test the hypothesis that NP-EGF-induced NO generation is a regulatory factor for c-Jun N-terminal kinase (JNK)-mediated apoptosis in BLBC cells.